Method for the production of dipicolinate

Abstract

The present invention relates to a novel method for the fermentative production of dipicolinate by cultivating a recombinant microorganism expressing an enzyme having dipicolinate synthetase activity. The present invention also relates to corresponding recombinant hosts, recombinant vectors, expression cassettes and nucleic acids suitable for preparing such hosts as well as a method of preparing polyester or polyamide copolymers making use of dipicolinate as obtained by fermentative production.

1 . A method for the fermentative production of dipicolinate, which method comprises the cultivation of a recombinant microorganism, which microorganism is derived from a parent microorganism having the ability to produce lysine via the diaminopimelate (DAP) pathway with L-2,3-dihydrodipicolinate as intermediary product, and wherein the enzyme aspartokinase in the lysine biosynthesis pathway is deregulated, and additionally having the ability to express heterologous dipicolinate synthetase, so that L-2,3-dihydrodipicolinate is converted into dipicolinic acid or a salt thereof. 
     
     
         2 . The method of  claim 1 , wherein said microorganism is a lysine producing bacterium. 
     
     
         3 . The method of  claim 2 , wherein said lysine producing bacterium is a coryneform bacterium. 
     
     
         4 . The method of  claim 3 , wherein the bacterium is a  Corynebacterium.    
     
     
         5 . The method of  claim 4 , wherein the bacterium is  Corynebacterium glutamicum.    
     
     
         6 . The method of  claim 1 , wherein said heterologous dipicolinate synthetase is of prokaryotic or eukaryotic origin. 
     
     
         7 . The method of  claim 6 , wherein said heterologous dipicolinate synthetase is from a bacterium of the genus  Bacillus , in particular from  Bacillus subtilis.    
     
     
         8 . The method of  claim 7 , wherein the heterologous dipicolinate synthetase comprises at least one alpha subunit having an amino acid sequence according to SEQ ID NO: 2 or a sequence having at least 80% identity thereto, and at least one beta subunit having an amino acid sequence according to SEQ ID NO: 3 or a sequence having at least 80% identity thereto. 
     
     
         9 . The method of  claim 1 , wherein the enzyme having dipicolinate synthetase activity is encoded by a nucleic acid sequence, which is adapted to the codon usage of said parent microorganism having the ability to produce lysine. 
     
     
         10 . The method of  claim 1 , wherein the enzyme having dipicolinate synthetase activity is encoded by a nucleic acid sequence comprising
 a) the spoVF gene sequence according to SEQ ID NO: 1, or   b) a synthetic spoVF gene sequence comprising a coding sequence essentially from residue 193 to residue 1691 according to SEQ ID NO: 4; or   c) a nucleotide sequence encoding a dipicolinate synthetase comprising at least one alpha subunit having the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 80% identity thereto, and at least one beta subunit having the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 80% identity thereto.   
     
     
         11 . The method of  claim 1 , wherein in said recombinant microorganism at least one further gene of the lysine biosynthesis pathway is deregulated. 
     
     
         12 . The method of  claim 11 , wherein said least one deregulated gene selected from aspartatesemialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose-6-phosphate dehydrogenase, transketolase, transaldolase, 6-phosphogluconolactonase, fructose 1,6-biphosphatase, homoserine dehydrogenase, phophoenolpyruvate carboxykinase, succinyl-CoA synthetase, methylmalonyl-CoA mutase, tetrahydrodipicolinate succinylase, succinyl-aminoketopimelate transaminase, succinyl-diaminopimelate desuccinylase, diaminopimelate epimerase, diaminopimelate dehydrogenase, and diaminopimelate decarboxylase. 
     
     
         13 . The method of  claim 1 , wherein the dipicolinate thus produced is isolated from the fermentation broth. 
     
     
         14 . A nucleic acid sequence comprising the coding sequence for a dipicolinate synthetase as defined in option b) of  claim 10 . 
     
     
         15 . An expression cassette, comprising at least one nucleic acid sequence as claimed in  claim 14 , which sequence is operatively linked to at least one regulatory nucleic acid sequence. 
     
     
         16 . A recombinant vector, comprising at least one expression cassette as claimed in  claim 15 . 
     
     
         17 . A prokaryotic or eukaryotic host, transformed with at least one vector as claimed in  claim 16 . 
     
     
         18 . The host of  claim 17 , selected from recombinant coryneform bacteria, especially a recombinant  Corynebacterium.    
     
     
         19 . The host of  claim 18 , which is recombinant  Corynebacterium glutamicum.    
     
     
         20 . A method of preparing a polymer, which method comprises
 a) preparing dipicolinate by the method of  claim 1 ;   b) isolating dipicolinate; and   c) polymerizing said dipicolinate with at least one further polyvalent copolymerizable co-monomer   
     
     
         21 . The method of  claim 20 , wherein said copolymerizable co-monomer is selected from polyols and polyamines and mixtures thereof.